Investigations of the tick burden on passeriform, water-associated and predatory birds reveal new tick–host associations and habitat-related factors of tick infestation

Background Previous studies on the tick infestation of birds in the Carpathian Basin focused on songbirds (Passeriformes). Thus, the primary aim of the present work was to extend the scope of previous studies, i.e. to include aquatic (water-associated) bird species in a similar context, especially considering that these birds are usually long-distance migrants. Methods Between March 2021 and August 2023, 11,919 birds representing 126 species were checked for the presence of ticks. From 352 birds belonging to 40 species, 905 ixodid ticks were collected. Tick species were identified morphologically and/or molecularly. Results Ticks from avian hosts belonged to seven species: Ixodes ricinus (n = 448), I. frontalis (n = 31), I. festai (n = 2), I. arboricola (n = 36), I. lividus (n = 4), Haemaphysalis concinna (n = 382) and Dermacentor reticulatus (n = 2). Nymphs of I. ricinus occurred with a single activity peak around March–May, whereas its larvae typically infested birds in May, June or July. By contrast, H. concinna usually had its activity maximum during the summer (nymphs in June–July, larvae later in July–August). Interestingly, two ornithophilic species, I. frontalis and I. arboricola, were most active around winter months (between October and April). A significantly lower ratio of aquatic birds was found tick-infested than songbirds. Several new tick–host associations were revealed, including I. ricinus from Greylag Goose (Anser anser) and D. reticulatus from Great Egret (Ardea alba) and Sedge Warbler (Acrocephalus schoenobaenus). Ticks were collected for the first time in Europe from two species of predatory birds as well as from Little Bittern (Ixobrychus minutus). Bird species typically inhabiting reedbeds were most frequently infested with H. concinna, and most ticks localized at their throat, as opposed to forest-dwelling avian hosts, on which I. ricinus predominated and ticks were more evenly distributed. Conclusions In the evaluated region, aquatic birds appear to be less important in tick dispersal than songbirds. However, newly revealed tick-host associations in this category attest to their hitherto neglected contribution. The results suggest that the habitat type will have significant impact not only on the species composition but also on the feeding location of ticks on birds. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-024-06229-1.


Background
Hard ticks (Acari: Ixodidae) affect animal and human life in several ways, among which the most important is that they are transmitters (vectors) of a broad range of pathogens with high veterinary-medical significance [1].Because each species of tick-borne pathogens has evolved to associate with certain tick vectors in which, as biological vectors, they can multiply and can reach the form infectious for susceptible vertebrate hosts, the local tick fauna will determine the endemic occurrence of tick-borne diseases [2].In this context, the highest contribution to local tick populations is probably achieved by birds, in terms of introducing new individuals and even new species [3].Their role in tick dispersal in urban areas is particularly high [4], especially when considering that in cities they outnumber medium to large mammals.Furthermore, birds are important carriers and distributors of ticks and thus of tick-borne pathogens, even on a transcontinental scale [5].
The history of investigating the tick infestation of birds in Central Europe and the Carpathian Basin goes back several decades [6].Most of these studies in the past were either based on random, sporadic tick collections [7] or annual removal of ticks from birds at one stop-over site [8,9].In 2022, however, a country-wide survey on ticks associated with avian hosts was also conducted [10].Nevertheless, all these bird tick studies focused on just one order of birds, the Passeriformes (songbirds).
Aquatic birds, for instance members of the order Anseriformes (ducks, geese, swans), are not known as hosts of ticks in Hungary [11] and were also rarely reported in this role in Europe [12].One of the underlying reason why anseriform birds appear to be neglected when studying avian tick hosts is probably the difficulty of sampling, i.e. they cannot be easily mist-netted and are rather funnel trapped or caught with other methods.At the same time, aquatic birds in general were reported to develop seroconversion to tick-borne encephalitis virus (TBEV) in Europe [13], and ducks in particular have been recently shown to develop viremia to this pathogen [14], justifying their epidemiological role.In addition, while geese are presumably main tick hosts and main vertebrate Borrelia reservoirs [15] and are long-known natural carriers of Ixodes ricinus-borne pathogens [16], during the past decades one of their most widespread species, the Greylag Goose (Anser anser) was not reported with tick infestation in Europe [12].
Thus, the aim of this study was to extend the scope of previous surveys on the role of birds as tick hosts in the Carpathian Basin, i.e. to investigate the following new aspects: (i) regions and habitat types previously not evaluated; (ii) avian hosts species which are rare or for other reasons were previously not examined; (iii) data on the anatomical location of tick infestation hitherto not analyzed in the context of avian traits influencing it; (iv) the tick burden of aquatic (water-associated) bird species, especially considering that these birds are usually longdistance migrants.

Sample collection and identification of tick species
During this study, 11,919 birds representing 126 species were checked for the presence of ticks.The samples were collected during bird ringing activities between March 2021 and August 2023 at 19 locations focusing on northwestern Hungary (Fig. 1).Songbirds (order Passeriformes) were captured with standard mist nets (Ecotone, Gdansk, Poland), which are 12 m long and 2.5 m high.Depending on the habitat, 1-12 nets were used.For water-associated birds from other orders (Anseriformes, Pelecaniformes, Gruiformes, Charadriiformes, Podicipediformes, Ciconiiformes), occasionally manual capture was also applied.In addition, rescued predatory birds (orders Accipitriformes, Falconiformes) were also included in the study.All birds were carefully inspected for the presence of ticks, starting from the throat and progressing by blowing the plumage through the beak, eyes and then ears.Ticks were removed from the birds with pointed tweezers and placed into pre-numbered tubes filled with 96% ethanol.Tick species were identified according to standard keys [17].In addition, the species identity of Dermacentor reticulatus nymph was confirmed molecularly based on the 16S rRNA gene (data not shown), as reported [10].

Statistical analysis
Prevalence data were compared with Fisher's exact test (https:// www.langs rud.com/ fisher.htm), and differences were considered significant if P < 0.05.Bird species were assigned into categories according to their typical habitat (Table 2; Additional file 1) as previously reported [10].

Species and developmental stages of ticks collected from birds
A total of 905 ticks belonging to the following seven species were collected from birds: I. ricinus

Seasonal distribution of ticks collected from birds
Due to the random distribution of sample collection days (Additional file 2), only larger monthly or seasonal trends were followed.Considering the two most frequently collected species, the occurrence maxima of the different stages are shown in Table 1.Thus, in the case of I. ricinus nymphs, there was typically a spring, March-to-May peak in their activity.The occurrence of larvae mostly showed a peak between May and July, which was also different between the years.The second most common species was H. concinna, for which the presence on birds in spring and autumn periods is seldom observed in Hungary (Table 1).The occurrence of nymphs of this species reached its maximum in June-July of each year; then, in July-August (typically by mid-August), this was taken over by the predominance of larvae.
The third most common species was I. frontalis, which occurred between October and April: larvae had their peak activity between October and December while nymphs between January and April (Table 1).
As an exception, a single nymph was found on a bird in August.The occurrence of females appeared to be random.Ixodes arboricola is the fourth species that was collected in larger numbers.Findings of this species were restricted to one place (Fig. 1: Halászi), where it was the most common tick species on birds on two ringing days in December.It was found from a total of 11 birds (n = 36), including larvae (n = 21), nymphs (n = 14) and females (n = 1).By contrast, in the relevant period, I. ricinus occurred on 10 birds (n = 12) and I. frontalis was collected from nine (n = 14).

Host associations of ticks collected from birds
The distributions of tick species and stages according to host species and bird orders are shown in Table 2.During the investigation, 352 individuals of 40 bird species were found to be infested with ticks.Water-associated avian hosts examined in this study (n = 1497) belonged to 53 species (data not shown) and six orders (see above).Among these, only five tick-infested individuals of three species were found (Table 2).This is significantly (P < 0.0001) fewer than in the case of songbirds (Passeriformes), among which 10,422 birds of 73 species were examined, and 340 individuals representing 32 species were tick-infested (Table 2).Ticks were also collected from four birds of prey (Table 2).2).Considering the rare hosts of I. frontalis, it was removed from a Fieldfare (Turdus pilaris), a Common Reed Bunting (Emberiza schoeniclus) and a Eurasian Tree Sparrow (Passer montanus) (Table 2).Ixodes Among the birds of prey (orders Falconiformes, Accipitriformes), tick infestation was observed in four species (Table 2).The highest number, a total of 19 ticks, were collected from the Western Marsh Harrier (Circus aeruginosus), of which two I. ricinus specimens were identified along with two H. concinna larvae and 15 nymphs.Of the remaining species (Common Kestrel: Falco tinnunculus, European Honey Buzzard: Pernis apivorus, Western Marsh Harrier: Circus aeruginosus, Eastern Imperial Eagle: Aquila heliaca), only nymphs of H. concinna were collected.
Among the birds associated with wetlands, tick infestation was detected among Greylag Goose (A. anser) chicks.Ticks were found on 37.5% of chicks (n = 3) < 2 weeks old, all of which belonged to I. ricinus (1 larva, 13 nymphs).Dermacentor reticulatus specimens were collected from birds on two occasions: for the first time in June (2021) a female tick from the tibiotarsotarsometatarsal joint of a Great Egret (A. alba) chick in the nest and for the second time in July (2021) a nymph was removed from a Sedge Warbler (A. schoenobaenus) near Lake Fehér (Fig. 2).In addition, we found the species H. concinna on Little Bittern (Ixobrychus minutus) and among galliform birds on Common Quail (Coturnix coturnix).

Species of ticks according to the feeding habitat of their avian hosts
Tick-infested bird species were also compared according to their typical habitat.Among bird species that are reed-associated (Table 2), infestation with H. concinna dominated (n = 293 H. concinna vs. n = 77 I. ricinus), while for members of the forest ecosystem (Table 2), infestation with I. ricinus was more typical (n = 29 H. concinna vs. n = 302 I. ricinus).This was a highly significant (P < 0.0001) difference.

Anatomic location of ticks on their avian hosts
The anatomical location occupied by ticks during blood sucking was examined for 100 birds, from which a total of 122 ticks were removed.Individuals of 18 bird species were included in this analysis.Most ticks were found in the throat region (n = 73), but they also occurred around the eyes (n = 15), in the corner of the beak (n = 24), in the ear canal (n = 6) and rarely in unusual places such as around the cloaca (n = 1), on the wing (n = 2) and on the top of the head (n = 1) (Fig. 3).Most ticks were collected from Savi's Warbler (L.luscinioides), in which we found ticks around the throat in all cases (n = 19).In the case of the Great Tit (P.major) and Common Nightingale (Luscinia megarhynchos), ticks occurred with the same frequency around the corner of the beak and the eyes as in the areas around the throat.Interestingly, the typical tick predilection sites differed according to reed-associated or forest-dwelling bird species.Among bird species common in reedbeds (Fig. 4), tick infestation of the throat area dominated (n = 54 ticks in the throat area vs. n = 16 ticks in other places), while for forest-dwelling bird species (Fig. 4), the localization was significantly (P = 0.0003) less frequent around the throat and was more evenly distributed (n = 13 throat area, n = 10 eye, n = 10 beak corner).

Discussion
During the 3 years of the study, mostly preadult developmental stages of seven tick species were collected from birds.This means that, with the exception of four species (two Hyalomma species, Haemaphysalis punctata and Dermacentor marginatus), all tick species reported so far from birds in Hungary were found [12].
The seasonality of different tick species on birds matched previous Central European results [7,[18][19][20][21].Accordingly, the spring peak of I. ricinus nymphs varied between the months of March and May during the 3 years of the study.The larvae reached their maximum level of infestation between May and July.During this study, I. ricinus was collected from several bird species, from which tick infestations had not been previously reported in Hungary or Europe [12].Thus, new hosts of this tick species in Hungary include the Common Firecrest (R. ignicapillus) and Eurasian Treecreeper (C.familiaris).In addition, I. ricinus was also found on a Greylag Goose (A. anser).This result is especially important not only because tick infestation of this bird species has not been previously reported in Europe [12], but anseriform birds are also long-known to play a role in the epidemiological cycle of I. ricinus-borne pathogens, including the tickborne encephalitis virus [13,14] and borreliae [15].
The second most common tick species was H. concinna.The number of nymphs reached its maximum in June-July of each year, and then gradually the larvae became more prevalent on birds in July-August.According to previous studies, the larvae are active from the end of May to October, while the nymphs occur from April to October, but as in the present survey, they were most common in the middle and end of summer [18,22].
Most bird species that were found infested with H. concinna in this study are known to be associated with reedbeds, similarly to the results of a long-term study on bird-associated ticks in Hungary [23].These hosts were already reported [7].In addition, for the first time in Europe, we have recorded H. concinna infestation on Common Kestrel (F.tinnunculus), European Honey Buzzard (P.apivorus), Western Marsh Harrier (C.aeruginosus), Eastern Imperial Eagle (A. heliaca) and Little Bittern (I.minutus) [12].Moreover, no ticks from the Western Marsh Harrier (C.aeruginosus), the Eastern Imperial Eagle (A. heliaca) and the Little Bittern (I.minutus) have ever been found in Europe [12].
The third most common species was I. frontalis, which almost exclusively occurred between October and April.This is in line with previous results when peak activity was observed between January and April, with rare collection days in August [7], similarly to this study.Larvae were more common between October and December while nymphs between March and April.The occurrence of adults showed a random distribution.In Western Europe (the UK), adults are the most common on birds in the winter months, while the occurrence of larvae and nymphs was not sharply separated and occurred at any time between March and October [24].These shifts are probably attributable to differences in the life cycle of I. frontalis under oceanic and continental climate in Europe.
Another ornithophilic tick species collected in this study is I. arboricola, which was previously found only a few times in Hungary [11,25,26].Interestingly, so far nothing has been established about its seasonality in Hungary, but according to a foreign publication, it is most common before breeding and in the autumn and winter periods, but it can be found on both chicks and adults during the breeding season [27].During this study, I. arboricola was collected on two occasions at the same ringing location, and both days were in December.
Ixodes festai was also found twice during this study, on Savi's Warbler (L.luscinioides) and on Dunnock (P.modularis), in both cases in the spring, on migrating birds.This tick is a less-known species with a southern distribution, which, according to our current knowledge, does not breed in Central Europe or Hungary, but is only brought by birds coming from the south, from the Mediterranean region in the spring [7].No one has previously reported this tick species from Savi's Warbler (L.luscinioides) in Europe [12].
The species I. lividus was also collected four times from Sand Martin (Riparia riparia).This is a hostspecific tick species; so far, it has only been described from Sand Martin (R. riparia) and once from Western House Martin (Delichon urbicum) [28].It shows a strong seasonality, so the females that were collected in this study also occur in the summer on nestlings and first-year birds [29].The hosts in this study represented the latter.
Dermacentor reticulatus has so far rarely been collected from birds in Europe [10,12].For the first time, it was found on a nymph from a Meadow Pipit (Anthus pratensis) [30] and for the second time from a larva from a European Robin (E.rubecula) [19].On the other hand, during our investigation, a female was found on a Great Egret (A. alba) at a heron colony and for the second time on a Sedge Warbler (A. schoenobaenus) in northwestern Hungary.Therefore, this tick species was collected for the first time from both bird species in Europe [12].In addition, no species of ticks have been previously reported from Great Egrets (A. alba) in Europe [12].This is the first study which demonstrated differences in the feeding location of ticks on birds according to the habitat characteristics of the latter.The most common place of tick attachment was in the throat region (60%), especially in reedbed habitats.Contrarily, in a study carried out at the Baltic coastline, 75.0% of ticks occurred in the corner of the beak, 14.6% around the eyes, and only 4.4% on the throat, 4.4% in the ears, 1.1% on the back or the top of the head and 0.5% on the inside of the corner of the beak [19].The most plausible explanation for the above differences between this and the Baltic study is that in the latter reed-associated bird species were underrepresented [19].Since the predilection site of throat region for tick location was shown here to be associated with reed habitat, the likely reason for this phenomenon is bill wiping [31], during which the throat will come into contact with the reed stem on which questing ticks are situated.

Conclusions
In conclusion, aquatic birds appear to be less important in tick dispersal than songbirds.However, newly revealed tick-host associations in this category attest to their hitherto neglected contribution, including the occurrence of I. ricinus on Greylag Goose (A. anser) and D. reticulatus on Great Egret (A. alba).For the first time to our knowledge, ticks were collected in Europe from two species of predatory birds as well as from Little Bittern (I.minutus).Bird species typically inhabiting reedbeds were most frequently infested with H. concinna, and most ticks localized at their throat, as opposed to forest-dwelling avian hosts, on which I. ricinus predominated and ticks were more evenly distributed on various body parts.Thus, the results suggest that the habitat type will influence not only on the species composition but also the feeding location of ticks on birds.

Fig. 1
Fig. 1 Map of Hungary showing the collection sites

Fig. 3
Fig. 3 Anatomical location of tick infestation among birds in this study: (A) below the eye in Common Starling (Sturnus vulgaris); (B) on the eyelid and on the vertex in European Greenfinch (Chloris chloris); (C) in the corner of beaks, margin of oral mucosa in Eurasian Blackbird (Turdus merula); (D) in the meatus auditorius in Eurasian Blue Tit (Cyanistes caeruleus); (E) below the eyes in Greylag Goose (Anser anser)

Fig. 4
Fig. 4 Number of ticks removed from 100 birds according to the anatomical location of tick infestation, species of avian hosts and their typical habitat

Table 1
Seasonal distribution of ticks and their developmental stages/sex, collected from birds in the study period (March 2021 to August 2023)

Table 2
Summary of tick species and developmental stages according to avian host taxa The third column (n =) shows the numbers of individuals of a bird species that were found tick-infested.Species codes (Additional file 1) of birds typically occurring in reedbed habitats are shown with superscript "1" and those with forest habitats with superscript "2" L larva, N nymph, F female, sp.species, Ia Ixodes arboricola, If Ixodes festai, Il Ixodes lividus, Dr Dermacentor reticulatus